SK2294A3 - Method of biological destruction of molluses - Google Patents

Abstract

PCT No. PCT/GB92/01248 Sec. 371 Date Mar. 8, 1994 Sec. 102(e) Date Mar. 8, 1994 PCT Filed Jul. 9, 1992 PCT Pub. No. WO93/00816 PCT Pub. Date Jan. 21, 1993.Phasmarhabditis nematodes for the biocontrol of agricultural and horticultural pests and more particularly to the biocontrol of molluscs. The nematodes are cultured with a nematode growth-promoting and pathogenicity-inducing bacterium and applied to the molluscs. The molluscs which are biocontrolled by the disclosed method include slugs, i.e. Deroceras reticulatum. In addition to the specific biocontrol of slugs the invention is applicable to the biocontrol of other molluscs, such as snails, i.e. Monacha cantiana, which are harmful to plants in the field or greenhouse, or to those which carry parasites harmful to humans or animals.

Description

Who is the bearer. liver flukes (lasciola hepaticaj and Bulinus species (family Eulir.idae) carrying Cpisthorchis sinensis. Ceiade Limacidae, Arionidae, MiIacidae and Helicidae belong to the order Stylommatophora. Family Bulinidae and Lymnae.idae belong to the Basommatophora order.

Current methods of controlling these pests are only partially effective and the available chemicals are highly toxic to birds and mammals. Therefore, there is a clear need to find more effective, lasting and less toxic methods of controlling shellfish.

SUMMARY OF THE INVENTION

It has now surprisingly been found that Nematoda roau Phasmarhabditis is an effective means of controlling a wide variety of molluscs. Among the species belonging to the genus Phasmarhabditis, the related organisms P. neopapillosa and P. hermaphrodita, which will be described in more detail below, are particularly effective. These species have been known for many years and are described in the literature and characterized mainly in Andrassy., 'A Taxonomic Reviev / of the Sub-'Jrder Rhabditina (Nematoda: Secernentina) fl98j, Orstom, Paris, France). However, the biological activity of these organisms against snail pests has not been known.

Accordingly, it is an object of the present invention to use the Phasmarhabditis species for controlling agricultural and horticultural pests harmful to human and animal health, in particular molluscs. These organisms can be harvested from slugs in the field and grown by the methods described below to obtain them in sufficient quantities for processing into suitable formulations that can be applied in the field or in a greenhouse. materials such as peat, clay and other solid or semi-solid carriers such as gelled materials. External tests, both on a small scale and on a small scale, have shown that nematodes can kill slugs and protect Chinese cabbage seeds and seeds or wheat seedlings from slugs at least as good or better than methiocarb, which is the best chemical for this purpose. currently available.

Biology of the organism

Nematodes were isolated from slugs collected at Long Ashton Research Station in the UK. It has been found that these nematodes cause a deadly disease of slugs, manifested by certain characteristic symptoms of which it is. the most noticeable swelling of the slug shell. These non-rotations have been found to be subordinate to fehabditin and a key has been used to further identify them (Andrassy, 1983). The two main taxonomic characteristics of this group of oral and male reproductive structures have been characterized by a nematode isolated at Long Ashton. } with isomorphic metastoma, and males, if present, have peladeran bursa, characteristic of the genus Phasmarhabditis.Andrassy (1983) noted two species that are morphologically identical to these nematodes, but differ from each other in the number of males present in these In Phasmarhabditis neopapillosa, males and females are equally numerous, while in Phasmarha'oiditis hermaphrodita males are extremely rare, it is not yet known whether P. hermaphrodita is a special species or only a biological variant of P. neopapillosa (Andrassy 1983). P. hermaphrodita was first described by Maupas in Archives de Žoologie (1900? · Vol. 8, pp. 46'4-624), which this nematode called Rhabditis caussaneli. He kept the cultures of this nematode on rotten meat for two years. He found that adult worms were predominantly protandric autogamous hermaphrodites. He further found that males are present only in very small numbers (L male per 1300 females) and that the number of males in cultures is not influenced by nutrient conditions. Maupas never recorded male-female copulation, which showed that there was no change in nematode fexundity or sexual offspring in the presence of males. Maupas did not regard this nematode as a slug parasite.

Phasmarnabditis neopapillosa boí described by Mengert, who called this nematode Rhabditis neopapillosa, in which the journal Zeitschriťt jur Morphoiogie und Oekologie 'líere (1953), Vol. 41, p. 311 to 349, concerning the relationship between nematodes in its study, concerns molluscs. He found this nematode in the form of resistant larval stages (called dauer larvae‘9 in the back of the digestive tract of the slug Limax cinereoniger).

Mengert considered the nematode P. neopapillosa to be a saprophyte that has been prospering on rotting matexia for many generations, but in spite of adverse conditions, young individuals do not mature and create resistant forms of dauer larvae that do not receive food. Mengert thought that? neopapillosa lives in the same way as two other species of Phasmarhaoditis papiliosa and P. hermaphrodita. He further believed that the dauer larvae of these three species would enter the body of the snails, if there was an opportunity, where they would remain in the form of dauer larvae until the slug died. Only then do they continue to develop. λ reproduce while feeding on the dead body of the snail. Mengert considered that the stay in the slug was not a necessary part of the nematode life cycle AIE assumed that the dauer larvae of these species actually have a certain stu5 in _t pen adaptation to life in the cochlea. He said, however, that these nematodes do not parasitize on slugs.

The nematode can be isolated from slugs harvested in the field using bait traps from crushed bran which are spread on. grassy area. By cutting the slugs, the nematode can be isolated from the digestive tract of the slug or mantle cavity. Many species of nematodes live in slugs (Mengert, 1953} and the identification of P. hermaphrodita and P. neopapillosa must be confirmed using a taxonomic key (Andrassy, 1933?). If only an infectious disease is found in the snail. Nematode stages fdauer larvae need nematode cultivated to identify them.

Nematoda Phasmarhabditis have been isolated at the Long Asht'on Research Station on many occasions. In some cases, the nematode population consisted of males and females, while in other cases the populations consisted only of hermaphrodites. Nematodes from populations of both types were examined by light microscopy and electron microscopy (SEM). Protein profiles in different populations were also determined after protein separation by isoelectric focusing. No differences between populations were found using any of the above methods. The isolated nematodes correspond to the available description of P. neopapillosa and P. hermaphrodita.

Nematoda Phasmarhabditis can be grown by the methods described in this description. It is already known in the art that nematodes that parasitize insects can be grown on a large scale for commercial use in liquid cultures using stirred tanks or so-called shells. Airlift trays with pieces of foam. Similar techniques can be used to produce P. hermaphrodita or P. neopapillosa in. v.eikom scale. Thus, the nematodes used in the present invention can be easily grown on kidney medium in the form of foam pieces or liquid culture, using techniques similar to those used to produce nematodes that parasitize insects. For the purposes of the present invention, it is recommended that the nematode culture be harvested in the dauer larvae state.

Requirements for attached bacteria

Nematoda Phasmarhabditis feed on bacteria. After isolation from dead slugs, it was found that the phasmarhabditis nematode is associated with many bacterial isolates.

the bacteria are able to promote good nematode growth and to compare the pathogenicity of nematodes reared on different types of bacteria.

the nematodes are preferably grown in cultures with a known attached bacterium (monoxenic cultures) and

bacterial species that are able to support the growth of

on a mixed microbial population, from slugs infected with slug-dead bacteria infected with nematodes.

The nematode can then be removed from all contaminating bacteria and transferred to cultures with a different single bacterial species. Incubation of these cultures allows the selection of bacterial isolates that are capable of promoting nematode growth.

Approximately 1ΌΟ of bacterial isolates were obtained from nematodes, slugs infected with nematodes infected with nematode-infected slugs, of which 15 were tested for their ability to promote nematode growth. Of these 15 isolates, a total of 9 isolates representing 8 species were found to promote good growth on agar. With the bacterial species that have been found to promote good nematode growth, the following are stated:

Pseudomonas fluórescens

Providencia rettgeri Serratia · Proteomaculans Aeromonas salmonicida Moraxella phenylpyruvica Bacillus cereus

Flavobacterium odoratum

Flavobacterium brevi

Capable of nematodes, grown on different types of bacteria, killed slugs, is possible, found by biological experiment. In such a biological experiment, slugs are exposed to a different number of nematodes and the resulting slug mortality is recorded. Using this method it is possible to quantitatively evaluate the pathogenicity (eg LD _C cJ nematodes against slugs and used this not the pathogenicity of nematodes grown in the ^ ii. It is important that the nematodes are specific bacteria, not only for the growth of the nematode (both in vitro and also for their ability to kill 'attached bacteria on entering the snail, Le deal with and rapidly reproduce nematodes, to the death of the snail.

Value for comparison. species supplied in conjunction because bacteria are important both in vivo) but mites. The nematode carries what it 's quick to do. /

Examples of suitable bacterial strains include Moraxella phenylpyruvica strain 48 and Pseudomonas fluorescens strain 141, samples of which were deposited in accordance with the Budapest Agreement in the National Collection of Industrial and Marine Bacteria, 23 St. Machar Drive, Aberdeen , AB2 IRY, UK) under accession numbers NCIMB 40508 and NCIMB 40509 on June 9, L992. The rseudomonas fluorescens 141 strain is a gram negative, oxidase positive, catalase positive bacterium that is immobile and shows a negative result in the O / F assay (Hugh and Leifson) in aerobic or anaerobic glucose cleavage. Moraxella phenylpyruvica 48 strain is a gram negative, oxidase positive, catalase positive bacterium that is. immobile · a Has a negative result in test 0 /? (Hugh and Leifson). The biochemical profiles of both strains on Standard substrates (API ZON test strip) are shown below.

Reaction of Moraxella Pseudomonas phenylpyruvica 48 fluorescens 141

NO ₃ -NO ₂ no ₃ -n ₂ indole glucose acid arginine dihydrolase ureaza esulin hydrolysis gelatin hydrolysis β-galactosidase

Assimilation of glucose arabinose mannose N-acetylgluko Zamine

maltose gluconate caprate adipate malate citrate phenylacetate

* = not tested / v

Usefully, variants of M. phenyplpyruvica 4.8 and P. fluorescens 141 are. can be obtained by repeatedly transferring pure cultures of these strains to subcultures. Variants can also be obtained by re-isolating Phasmarhabditis nematode bacteria that were previously grown in conjunction with any of these Tribes, or by re-isolating nematode-infected snail bacteria. Such variants may have. grounded genotypic or phenotypic changes due to environmental or selective pressure. Useful derivatives of M. phenylp.yruvica 4S and Kenya r. fluorescens 141 can be constructed by introducing DNA from another organism that encodes the desired attributes. Methods for introducing foreign DNA into bacteria are well known to those skilled in the art, and include techniques such as plasmid transfer, transduction, and transfection. Useful mutants of M. phenylpyruvica 48 and P. fluorescens 141 can be obtained by mutagenesis using methods well known to those skilled in the art, such as chemical techniques (e.g., using nitrosoguanidine), physical techniques (e.g., using ultraviolet light). and genetic methods (e.g., transposon mutagenesis). Such variants, derivatives, and mutants of these strains may be varied as regards properties such as growth rate or the ability to grow on certain food sources, but retain in particular the properties that are important to the present invention, i.e. the ability to promote

For use in controlling agricultural pests, nematodes are collected from the fermenters by centrifugation, filtration or removal of the components of the spent medium and either processed directly into a pesticidal composition or stored in the form of chilled aerated aqueous suspensions before being processed into that composition. For semi-agricultural use, the nematode may be formulated into aqueous suspensions on solid carriers such as activated carbon, clay, peat, vermiculite or polyether-polyurethane sponge, or encapsulated in gels such as an alginate or polyacrylamide gel. A particularly suitable composition is a composition comprising dried or partially dried nematode. The nematode composition can be applied in pest control in the form of an aqueous suspension which is made from it and which is applied to the area to be treated, for example by spraying, irrigation or pickling.

The invention is illustrated by the following Examples. These examples are illustrative only and do not limit the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

isolation from Phasmarhabditis

Live nematodes obtained from slugs in the field harvested using traps with crushed bran, such as ^- bait is placed on Iadvinove agar medium made by mixing 10% homogenised pig Íadvín, 3.5% corn alley, 2% agar and 84.5% water weight) which was autoclaved and poured into petri dishes. This medium stimulates the growth of bacteria associated with nematodes. The nematode feeds on these bacteria and grows and reproduces on the dishes.

Example 1 a d 2

Isolation of nematode-associated bacteria or nematode-infected snails

Bacteria associated with nematodes or slugs infected with nematodes can be isolated by any of the following methods:

Isolation of bacteria from nematodes

The nematodes are surface sterilized by immersion in a sputter of ethylmercuritiosalicylate sodium (Thimerosal) at a concentration of 1 g / L for 1 hour and then transferred to fresh Thimercsal solution for a further 3 hours. Bacteria can be released from nematodes by the following 2 sterile microbiological techniques:

a) The individual nematode larvae are transferred to a drop of sterile sodium chloride solution, which is placed on a flame-sterilized microscope slide. The nematodes are then cut at the mecolecular sites along the length of their body. A drop of sodium chloride solution containing nematode dead bodies is then transferred to a 9 cm diameter petri dish with a sterile Pasteur pipette and spread on the surface with a spatula that is coxa sterilized in an alcohol burner flame.

b /

A large amount of surface sterilized nematodes is suspended in 1 ml of sterile Hinger's solution and the resulting suspension is transferred to 5 ml of a Teflon tissue homogenizer. The nematode suspension is crushed and then transferred to 9 ml of sterile broth. The broth is shaken vigorously and serial dilution is performed. 0.1 ml aliquots j. samples of each diluted soil are placed on a nutrient agar plate, spread over the surface with a glass spatula and incubated. After 48 hours incubation at 25 ° C, different bacterial isolates and blunt transfer can be selected based on colony morphology ·

insulation

with the onemic alcohol of prosperous xenic cultures, pieces of foam containing nymphs are removed with pliers which have been sterilized in the flame of the ratchet. Each piece is placed in a test tube containing 2 ml aliquots of different sample samples. spread on nutrient agar plate, then incubate.

iii Isolation of bacteria from live snails infected with nematodes

p. hermaphrodita / P. neopapillosis infects the mantle area of the slug, where it also reproduces. just

bacteria may have been isolated from this area. The comb is first wiped with a dry wad of cotton wool to remove the slime as much as possible. Thereafter, the surface of the comb is wiped with 70% (v / v) ethanol for surface sterilization of the comb. The flame-sterilized needle is punctured by the sheath, and then droplets of liquid at the end of the needle are transferred directly to nutrient agar plates, spread with a glass spatula and incubated.

iv Isolation of bacteria from dead slugs

The tissue smears from the carcass of a slug that has died after nematode infection and are coated with nematodes are suspended in the broth using a bacteriological tail. Serial dilutions of the resulting suspension are made and a 0.1 ml aliquot is spread on nutrient agar plates and incubated.

Example 'J

A method of securing bacteria that promote the growth of nematodes.

The female reproductive tract of nematodes is usually sterile (Poinar and Hansen, Helminthological Abstracts, Serie S, (1886j vol. 55, no. J, pp. 61-81)) and therefore juveniles (Jl) are sterile immediately after hatching. pregnant maternal nematodes, taken from nematode or snail cultures, are transferred to a sterile watch glass containing 0.2 g / l Thimerosal solution and kept overnight at 10 ° C. During this time, adult eggs are hatched and juveniles are released (Jl) The next day juveniles are transferred by pipette to centrifugation

new strength containing 500 U / ml penicillin G and streptomycin suifate. The juveniles are left in this solution for 24 hours at 10 ° C. Concentrate each year by gentle centrifugation (5 x g, for 10 minutes), collect from the bottom of the tube and resuspend in a sterile quarter-sterile Ringer's solution and centrifuge again. Resuspension and centrifugation is repeated one more time to remove all traces of antibiotic. The larvae are then placed on a sterile watch glass. The nematodes can then be handled individually using a micropipette which is made by stretching a dropping pipette in a Sunsen burner up to a width of approximately 0.1 mm. The nematode cultures are grown on Glacial agar (described in Example L) in J cm petri dishes. A batch of one bacteriological seed of a 1 hour old culture medium with cultured bacteria is plated line-wise across half of the JO mm agar plates. 10 scenic juvenile nematodes, obtained as described in Example 8, are placed on the edge of the non-bacterial half of the retri dish so that the nematode has to cover at least 15 mm distance across the non-bacterial surface before it reaches the xu of the test bacterium. Plates are incubated at i5 ° C.

Any bacterium that is present on the namaxodas and that has not been killed during the axenization process creates visible colonies in this half of the dish, and such misxies may then be discarded.

After 1 week, dishes showing bacterial contamination are discarded in the clean half. After 2 weeks, the number of nematodes present on the dish can be counted by direct microscopic observation; the lid of the petri dish is removed and replaced with another one on which the counting grid has been pre-marked. After J weeks, the nematode can be counted again by flushing the nematode from the agar to a known volume of water and counting the nematode in the resulting slurry in Peter's 1 mm counting chamber.

Nine different types of bacteria that have been isolated by the methods described above are tested for their ability to promote nematode growth. the results are summarized in Table L.

Tabuíkai

The number of phasmarhabditis nematodes in the Petri dish after 2 and 3 weeks of growth in a monoxenic kit with different bacteria. In the statistical analysis, the data are converted to Logarithms

bacteria Mon 2 weeks PO 3 weeks Count Log Count Lo, axenic 2 0.41 0 0.00 Bacteria 1A 170 2.18 18090 4.22 Bacteria 17 0 0.04 0 0.00 Bacteria 34 1 0.13 890 1.00 Bacteria 48 60 1.70 54060 4.73 Bacteria 54 80 1.53 25950 4.39 Bacteria 1160 3.06 86340 4.93 FROM Bacteria 83 520 2.46 67000 4.78 Bacteria 141 690 2.77 75220 4.85 Bacteria 156 250 2.26 83630 4.89

The standard deviation for comparing the logarithms of the number of nematodes is 0.204, 12S L.F.

After 3 weeks, highly significant (P <0.001) differences in the ability of bacteria to promote nematode growth occurred.

Method of mass cultivation of Äiasmarhabditis nematodes in a culture with pieces of foam

Nematodes can be mass grown on pieces of polyether-polyurethane foam using similar techniques to those developed for the mass production of insect-parasitic nematodes (Bedding, in Nematologica (1981), vol. 27, pp. 109-114 and Annals by Applied Biolcgy) (1984), vol 104, pp. 117-120. This medium consists of 65% pork laden, 15 phi of bovine fat and 25 ·% water (% by weight). The baking fat is melted in a wide pan above a circular gas burner, then added to the kidney homogenate, which is thoroughly mixed with the fat and cooked until brown until it is brown. Waring, where the pieces are grinded again, and the resulting mixture is then blended with 12 parts by weight added to one part by weight of the foam pieces. Anchovies or autoclave bags described by Sedding (1984) Cultures with pieces of foam are simultaneously inoculated with both namatods and bacteria mi. In each bag, slit at the top and cut 75 ml of overnight bacteria. The bacterial culture may be in the form of a mixed microbial culture obtained as described in Example 2, or in the form of a pure bacterial strain selected on the basis of its ability to promote nematode growth as described in Example 3.

Add nematodes on agar from Petri dishes or foam kicks from previous bag cultures. The bag tours are incubated at 15 ° C ρο for 3 weeks, as many infectious juices can be seen inside the bags, leaving the spent media in the bags. Nematodes are collected from the Pieces of Foam by a modified funnel extraction procedure similar to that used to isolate nematodes from ground samples. A 17.5 cm diameter copper sieve is covered with a 17.5 cm milk filter and placed on a 50 cm dish under a flower pot. The pieces of bag foam are placed in sieves to a depth of approximately 2 cm and then the pots are filled with water so that the water level just touches the bottom layer of the pieces of foam. The sieves are then kept in this state overnight. During this time, the living nematode is passed through the milk filter and collected in the water under the filter. Cleaning the nematode suspension of the spent medium, and ^the bacteria it with several changes of water 3 then the nematodes are stored in aerated water at 10 DEG C. until the time of use ';'

eg 5 Liquid Culture monoxenic nematodes Fhasmarhabaitis Axenizované The nematode is grown on a solid medium with containing kidney with the relevant bacteria. in three weeks

the nematode is transferred to a liquid culture.

The nematode is cultivated in a culture placed in the sword fossa under the following conditions:

Kidney medium 1% yeast extract, corn oil

Container: 2 50 ml conical flask with 50 ml medium

Temperature: L5 ° 0

Shaker rotating frequency: 200 min

The flasks are inoculated with 1 mu of bektsria grown in culture medium. After 24 hours, the nematode is flushed into the flasks with sterile tap water followed by a three week incubation.

The nematodes are rinsed twice with sterile water and counted to use the nematodes as an inoculum for experimental cultures. The nematodes are added to flasks pre-inoculated with bacteria at 3000 nematons per liter of flask contents.

Nematodes are grown in conjunction with 4 different types of bacteria. During the cultivation, nematode numbers are detected at different times. Dauer larvae stage, also known as infectious juvenia, is considered to be a nematode with a second degree of cuticula preserved.

The nematode is counted after 20 days of incubation and the results are shown in Table 2.

T a b u k a 2

Liquid culture of monoxenic nematodes

Bacteria year Mean number of nematodes in 1 m ± replication Dauer narvae left stage

P. fluorescens 6 1220 110 with. proteomaculans 6 11500 7400 P. rettgeri 6 99900 189000 M. phenylpyruvi 3 72000 223000

Those skilled in the art. based on the conditions described in this example, they can easily solve mass production of nematodes in liquid culture in large-scale fermenters.

- 19 example. 6

Method for selecting bacteria conferring pathogenicity against slugs

Nematodes grown in a monoxenic culture with 2 species of bacteria, Providencia rettgeri and Moraxella phenylpyruvica (see Example 5), were tested for pathogenicity against Deroceras reticulatum. A plastic box (dimensions 135 x 75 x 50 mm) is filled with 440 g of air-dried earth aggregates with a diameter of 12.5 to 25 mu, obtained by sieving. The seed aggregates from each box are removed and soaked in water.

First, untreated boxes are used in which no nematode is added, and Shatsui to which are added a non-soda in 5 different doses of 15000, 2J000, .35000, 55000, and 75000 nematodes per plastic box. For all 5 experiments with both batches of scnoxic nematodes, 2 boxes (biaxial replication) were used.

The nematodes are counted and their respective number. is suspended in 50 ml of tap water. The aggregates are repositioned and the nematode suspension is evenly distributed over the aggregate surface, layer by layer, 10 individuals of D. reticulatum are placed in the middle layer in each box. On aggregates in each crab! is evenly distributed. 50 mL tap water without adding any other nematode so that the resulting moisture content in each box is approximately 30% by weight.

Slugs are grown in soil for an infection period of 5 days at 10 ° C 3, then removed and transferred to Petri dishes, where they are grown individually and fed with discs taken from Chinese cabbage leaves. After another 9 days at 10 ° C (14 days after the initial nematode exposure), the metal was recorded. The mortality data is entered, which is evident from the mortality data, plotted to nematodes i.e. nematodes, / hikes with both. bacterial count of dead and live slugs due to mortality of untreated cathodes. Corrected.

In the experiment, the nematode cultured c. rettgeri pathogenic to D. reticulatum. This method can be used to sexify other strains of bacteria, such as o. fluorescence 141, which induces pathogenicity to slugs.

Example 7

Processing of Phasmarhabditis nematodes into pesticide pros' grades

The monoxenic nematode (Phasmarhabditis), which has been found in the ice, is associated with the strain 4S II.

phenylpyruvica (see example, which is carried out for as long as the nematode does not get rid of residual growth medium. Wash the nematode * by centrifugation to a water growth formed by nematodes, which contains 0.1 x 10 ^- to 2.0 The nematode paste is mixed with a clay based on calcium montmorillonite to form a water dispersible powder mixture containing 0.05 x 10 ⁶ to 1. S x 10 6 nematodes per gram (wet weight).

The ability of Phasmsrhabditis nematodes, produced in culture from pieces of foam, to kill various species of slugs

Namatoda Phasmarhabditis, which boxed ⁷ on a mixed bacterial flora by the methods described in the example. 4 were tested in biological poison against six harmful slug species. These species are Derocereas recitulatum, D. caruanae, Arion ater, A. intermedius, A. distinc tus and Tandonia (Milax) soweroyi. Snails were collected into traps with ground bran bait at Long Ashton Research Station in November

All snails were grown, except A. form (770 mg mean weight) _{of the} juvenil nematoda were grown in xenic bag cultures with pieces of foam as described in Example 4. In plastic boxes with dimensions of 1J5 x 75 x 50 mm was placed 440 g of air-dried, ground-to-grain aggregates with a diameter of 12.5 to mm, which were obtained by sieving. Orioliz 5 was added to each nematode-treated box

1.9 x 1.0 Phasmarhabditis infectious larvae, suspended in 130 ml tap water. The untreated boxes were made of them

r.ematôd. Into each of the except 130 ml cruprided. tap water free _of the skate was charged with 10 slugs, hov T. sowerbyi and A. ater which were added coio each fifth

A. distinctus snails were treated with nematodes and x8 salivated but untreated populations as control animals. In all other species, 20 snails were treated and 20 snails were left untreated as control animals. The snails were left in the soil for a pathogenic infectious period, after which the soil boxes were disassembled and the number of dead snails was recorded. The surviving snails were transferred to 9 cm petri dishes, lined inside with fixed tissue paper, where they were kept neat and fed with leaf rolls of Chinese cabbage. Soil boxes and petri dishes were kept at ± 0 ° 0 throughout the duration of the biological experiment. The number of dead slugs was recorded twice more at three-day intervals.

that of the individual snail species in the treated and untreated boxes at each moment, when the recording was made, was compared using a hair test. The results are shown in Table J.

¾ a b u k a 3

Percent mortality of different slug species at 5, 3 and 11 days after nematode treatment or untreated

Kind of slug eye ^- Frosted days untreated After treatment. days off. After 11 days treated. untreated Deroceras reticulatum 100 10 100 25 100 40 Deroceras caruanae 70 10 100 15 100 20 Aion ater 5 0 40 0 100 0 Arion intermedius 100 40 100 60 100 70 Arion distinctus 6 6 88 11 100 28 Tandonia sowerbyi 20 15 80 15 100 25

For a pathogenic infectious period, the mortality differences between nematode-treated slugs and untreated slugs were highly significant (<&_lt; Q _t QOi) ₉ for the 2nd reticuiatum, D. caruanae and A. intermedius. Mortality differences between treated and untreated snails in the other three species were not significant at this stage. After eight days, the mortality differences between treated and untreated snails were significant. for all species tested (p <U.D01) for D. reticuiatum, D. caruanae, T. soweberyi and A. distinctus with Ρ <ΰ.ϋ1 for A. atter and A. intermedius. By day 11, all slugs on which nematode had been applied had died. Mortality differences between treated and untreated snails were significant for all species (? <0.01) for A. intermedius and F <U.CQ1, for all other species). The difference in A. intermedius was not so great because many untreated ä * ·

Ä, .kov has died

From these results it is obvious that they are capable of nematode phasmarhaskasané species of slugs.

Capability of nematodes

Phasmarhabditis, grown in kuxtures with pieces of foam, reduce plant damage caused by the snail Deroceras reticuiatum under different conditions

An attempt was made on mini-fields to compare damage to Chinese cabbage seedlings by slugs on untreated, treated areas.

aathiocarou (considered to be the best available slugs) and shelves of ducks treated with a single high dose of nematodes produced in a foamed piece kit on a mixed bacterial flora as described in Example J. A series of 40 micro-fields containing hiine earth on the substrate were used. of the rough grain. The dimensions of the boxes were 70 χ 70 and their box depth was 30 cm. Boxes separated from each other. wooden or concrete hoops, on which the box was mounted a 13 cm high fence of wire mesh with a diameter of 0, S mm, which represented a barrier to the slug's movement between the boxes.

Between March and June 1989, 36 of these fields were populated with slugs. Not added to the remaining four fields. 'slug slugs and these boxes served as a measure of the resident slug population. 5 ha of harvested el. D. reticuxatum were placed in each of the controlled fields. These snails were kept for at least two weeks in quarantine boxes to ensure that they did not carry any parasites. Each field was inserted during the three-month period, 34 in the laboratory kept newly hatched D. reticulatum, so that at the beginning of the experiment, slugs were present in the fields at many stages of development.

The experiment was conceived by a dot-i randomized block, 2 untreated boxes, 1 tax box that 9 replicates were made each block including * nematode-treated and 1 box treated with methiocarb palettes.

In 300 ml of tap water 1.05 x nematodes 3 were suscered. Another 100 ml of tap water was used to flush the water and was used to rinse the kettle and the water was poured into the boxes, the untreated boxes and the poxies treated with ncthiocarb. 1 l of tap water, methiocarb relays were wished at the recommended dose for field treatment (5.5 kg / ha = 0.275g / field). The pellets were weighed and hand-spaced evenly across the fields. There were plots throughout the experiment

- 25 irrigated from above by means of irrigation pipes to ensure favorable conditions for the activity of snails.

At the beginning of the experiment, there were planted seedlings (square 3 x 3) of Chinese cabbage grown in a greenhouse for each field. The plants were controlled ^{from the} 2 times a week and an estimate (with an accuracy 5%) were detected damage to plants slugs.

were ane

Oh.

Two weeks after planting, the seedlings were completely, destroyed in some untreated fields, leaving the remnants of the old seedlings of them all. these boxes are removed and new ones have been planted. This was repeated after an additional 2 weeks. After another 2 weeks, the experiment was terminated (total duration of the experiment was about weeks). Seedling damage during the experiment was recorded twice a week. Copper-braided plotixes between poxies were released in one of the blocks (block #?) After the first 4 weeks, allowing slugs to move between these fields. These fields were therefore ignored and the results recorded after 5 3 6 weeks relate to xen .ts δ blox.

On each poxus box from the box, 2 soil samples (25 x 25 x 10 cm) were taken from the remaining S blocks, with 1 box taken from the middle and 1 from the southeast corner of each box, samples were sequentially irrigated for $ days in LäRS Snail Extraction Unit (Glen XWiltshire, Proceedings 1986 British Tropicrotection Conference (198c), SV · 1, pp. 139-144) and snails were removed from the surface every day.

The extent of slug seedlings damage at each treatment during poxus is apparent from FIG. '1.

Variation analysis. after angular transformation, in order to stabilize the variation, it shows that both the pellets by the methio line and the non-stably (<C, OO1) pellets decreased by the seedlings. In the first subtraction (4 weeks after treatment), more extensive damage was found to the nematode-treated patches compared to the methiocarb-treated patches, but when the seedlings were growing, they overcame the initial hop. At the end of the first week, nematode-treated fields showed less damage than methiocarb-treated fields, but this difference was not sintering. significantly (p &lt; .05) lower damage, compared to methiocarb-treated fields and this status of 0, C1) remained recurrent until the end of the year.

: t r »were ov,

Deroceres' Q +11 TI

- “‘ - J λ ·· *. · *% Ä * «« m r u z-n

Hoet — 'erid lôr' has a key oravcepadobically introduced sometimes for ν 'h-m axis.

med ‘J / 3, b

Exercise is required, not for 3 weeks of exposure to corn alteration, similar to a leaf that is borazornazus naive.

metal in the described experiment

In earth samples from 4 poxic? _eticuiatum, slugs on the other. This does not mean that ns xtore were not diminishing or shading the difference of ca. 3. paxlens there were no slugs of D. reticuiatum on the patches or there was little play

Celkove ✓

: 5L :: ov xtrsnov dr>.

The square root, n leading, are apparent from FIG. 2 • J υ u.

ic> cej sne j.;. ·. ;;

These results were significantly less nematode treated slugs than z (p <0.01) for all species of snails extracted. ..

untreated patches and for the species of non-muslims as well as those of untreated pops, and for all the slugs and reticmatus V, although the slugs were extracted from the methiocarb-treated nematode patches, this reticuloid difference is also bomo treated xÍcok (PC, 05 t type ¾)

00. "

D.

ne ooi sxg c r.

oaller with carb, I was not podsi despite the fact that rn.eth.io r * · ´ *> · ί -s * and * • -V ** ·· W. - - JJ lens eye has a smell that-

killing various, pest species from

In contrast to the m-S M · strain as described in Example 5, the boxes used in the biological experiment against various species of molluscs, including. Monacha cantiana, as described in the Example. The results are shown in Table 4.

8th

'P 3 c> - <X iC 3 .1 Percentual plague tolita róznyc h urumov harmful molluscs after cet monoxenium okay nemátón ol EBO without treatment Species Trven ie biology- After treatment with Dez treatment. molluscs the experiment (days) Deroceras reticulatum 11 100 15 Deroceras caruanae 11 100 10 Monacha cantiana 5 100 0 Arion intermedius 5 100 0 Arion distinctus 11 100 0 Tandonia sowerbyi 11 70 5

+ And differences in moi bditis sp., Monoxenized. with 48 M strain

The spectrum of activity of the non-oxenal nematodes was not changed by the elimination of pyru * T * y ^- * C, -

not in comparison to xenic ema:

Phasmarhabditis β-onoxenic nematodes were grown in conjunction with M. phenylpyruvica or P. rettg-ery in the manner described in Example 5 and used at various doses in a biological rat against the slugs of Q. reticu.atum.

in the manner described in Example 8. The results are summarized in FIG. J. Both types of monoxemic nematodes were active against

D. reticulatum.

Example 11

The ability of monoxenic nematodes Phasmarhabditis suppressed plant damage caused by the snail Deroceras reticulatum under field conditions

A poxic attempt was made to compare the damage to winter wheat (Lercia variety) caused by slugs, untreated poxies, fields treated with methiocarb pellets and fields treated with different doses of nematodes. Monoxenic nematode boxi grown in conjunction with strain 48 of M. phenylpyruvica, as described in Example 5 and as described in Example 7, treated with clay to disperse a water-dispersible powder containing 0.26 x 10 6 nematodes in the gram of the resulting composition (wet weight) ). The nematodes were applied immediately after sowing the fields in the form of a water spray with a volume equivalent to 1100 l / ha. Methiocarb pellets were applied by hand at the recommended starting dose of 5.5 kg / ha.

Surface traps and ground samples were used to monitor the slug population in the test fields. Many different slug species have been found in the bclo plots, including Leroceras reticulatum, Arion silvaticus, Arion subfuscus, Arion ater, Tandonia soweberyi and Milax gagates, but D. reticulatum clearly prevailed.

Six weeks after seeding, the fields were checked for seedlings, seedlings. This control represents an estimate of lethal damage to slugs (i.e., a reduction in the number of emerging plants). At the same time, an estimation of sub-lethal worms of snails (i.e., eat plants by snails) was made, based on a visual estimate of randomly selected plants. The mean number of emerging wheat plants, based on 0.5 m of furrow length in various treatments, is shown in Table 5.

Mean number of emerging wheat plants, based on furrow length, at various treatments in the poin test (evaluation done 6 weeks after sowing)

Treatment Mean number of emerging plants without treatment 12.93 dose nematodes 1 x 10 ⁸ / ha 12.78 dose nematodes 3 x 10 ⁸ / ha 13.95 dose nematodes · 1 x 10 ⁹ / ha 13.25 dose nematodes 3 x 10 ⁹ / ha 14.88 nematod dose 1 x 10 ¹⁰ / ha 16.50 methiocarb 14.57 standard deviation = 1.314 (399 df)

As the dose of nematodes increases, the number of emerging plants is clearly increasing, indicating that nematode treatment has reduced lethal damage to slugs.

Data relating to the mean pecental area of the leaf damaged by the snails was converted to un prior to analysis. The results are shown in Table 6.

- 31 T a b u k a 6

Mean angular percentage area of leaf damaged by slugs, based on the plant at various treatments in the poin test (evaluation is 6 weeks after sowing)

treatment

Mean angular percentage area of leaves damaged per plant

without treatment 31,82 dose of nematodes 1 x 10 ⁸ / ha ...... ........ 29, .15 nematode dose of 3 x 10 ⁸ / ha 28.87 nematode dose of 1 x 10 ⁹ / ha 22,11 nematode dose of 3 x 10 ⁹ / ha 18.63 nematode dose of 1 x 10 ¹⁰ / ha 16.49 methiocarb 25.17

standard deviation = 3,395 »(24« f.)

Substantial differences in the slug-damaged leaf area between treatments were found (P < 10 < 0 &gt; O1 &lt; -1 &gt;), with plants treated with the three highest doses of nematodes showing significantly (P &lt; Plants from the highest dose of nematodes treated showed significantly (P ^ 0.05) less slug damage compared to methi line treated plants. Thus, it is clear that nematodes are able to provide a good level of suppression of sub-lethal damage caused by slugs.

12

The ability of the monoxic nematodes Phasmarhabditis to kill water slugs of Lymnaea stagnalis

Monpxenic nematodes were grown in conjunction with 48 M. phenylpyruvica strain, as described in Example 5, as described in Example 7, using clay to form a highly dispersible powder containing 0.36 x 10 ° nematodes in the gram composition (wet weight) . Ten specimens of llaminous stagnalis were added to each of the five clean tanks of fish, which were half-filled with pond water containing some aquatic plants as a food source for the snails. The tanks were aerated with a small air pump and kept at 15 ° C.

Approximately 6 x 10 ° nematodes were added to each of the four tanks in the form of a water dispersible composition. No nematode was added to the fifth tank, which served as a control. After 3 days of incubation, the mean mortality of snails in nematode-treated tanks was 45 and increased to 100% after 6 days. For 6 days of incubation, no mortality was recorded in the untreated control tank.

?! /

Claims (27)

PATENT CLAIMS 1. Use of Phasmarhabditis nematodes for the control of agricultural, horticultural and pests threatening human and animal health. Use according to claim 1, wherein the nematode is selected from the group consisting of P. neopapillosa and P. hermaphrodita. Use according to claim 1 or 2 in conjunction with a suitable growth promoting bacterium. Use according to claim 1 or 2 in conjunction with a suitable growth promoting community. Use according to claim 1, 2, 3 or 4 for controlling mollusc pests in agriculture and horticulture. Use according to claim 5 for controlling harmful slugs of the family Limacidae, in particular Deroceras reticulatum and Deroceras caruanae. Use of the stage of claim 5 for controlling harmful slugs from the family. Arionidae, in particular Arion ater, Arion intermedius and Arion distinctus. Use according to claim 5 for controlling harmful slugs of the Milacidae family, in particular Tandonia sowerbyi. 9 »Use according to claim? for controlling harmful slugs of the family Helicidae, in particular Monacha cantiana. Use according to claim 1, 2, J or 4 for the control of mollusc-like pests endangering human and animal health. Use according to claim 10 for the control of slug pests of the genus Lymnea. Use of a stage according to any of the preceding claims, wherein the nematode is applied in the form of a dauer larvae. Use according to any one of the preceding claims, wherein the nematodes are applied in conjunction with bacteria that promote the growth of these nematodes. 14. Strain 48 of Moraxella phenylpyruvica, a sample of which has been deposited under accession number NCIMB 40508, or a variant, derivative or mutant thereof, having the ability to promote growth and induce pathogenicity of Phasmarhabditis nematodes against molluscs. A strain 141 of Pseudomonas fluorescens, a sample of which has been deposited under accession number NCIMB 40509, or a variant, derivative or mutant thereof, having the ability to promote growth and to induce pathogenicity of phasmarhabditis nematodes towards molluscs. 16. An agricultural and horticultural mollusc pest and a mollusc pest endangering human and animal health, comprising a Phasmarhabditis nematode species, in association with a suitable bacterium or bacterial community, promoting the growth of these nematodes, and a suitable carrier; or encapsulating agent. · »»> .. H _κ ; L ».U_T. ''>' · »..... Composition according to claim 16, characterized in that the nematode is present in the form of a dauer larvae 18. A composition according to claim 16 comprising a nematode species of P. neopapillosa or P. hermaphrodita. 19. The composition of claim 16, wherein the growth promoting bacteria is selected from the group consisting of: Pseudomonas fluórescens Providencia rettgeri Serratia proteomaculans Aeromonas salmonicida Moraxella phenylpyruvica Bacillus cereus Flavobacterium odoratum Flavobacterium brevi. 20. A composition according to claim 19, wherein said growth promoting bacterium is Moraxella phenylpyruvica strain NCIMB 40508 or Pseudomonas fluorescens NCIMB 40509. 21. The composition of claim 16, wherein the carrier is a clay. A water-dispersible powder composition according to any one of claims 16 to 21, comprising a calcium montmorillonite clay, water and nematode, wherein the nematode concentration is in the range of 0.1 x 10 -2 to 2.0. x 10?, preferably from 0.3 x 10? to 0.8 x 10? per gram wet weight. 23. A method for producing shellfish nematodes, characterized in that the Phas' marhabditis nematode is grown in a liquid medium, wherein the growth medium is pre-inoculated with at least one growth-promoting bacterium and inducing nematode pathogenicity, and then the nematode is collected in dauer larvae form. 24. The method of claim 23 wherein said growth promoting bacteria is selected from the group consisting of: Pseudomonas fluorescens Providencia rettgeri Serratia proteomaculans Aercnnonas salmonicida Moraxella phenylpyruvica Bacillus cereus Flavobacterium odoratum Flavobacterium brevi, 25. The method of claim 24, wherein the growth promoting bacterium is either. Moraxella phenylpyruvica or Pseudomonas fluorescens. 26. A method according to any one of claims 23 to 25, wherein the growth medium comprises a source of vitamins and minerals, a source of triglycerides, and a source of protein. 27. A method according to any one of claims 23 to 26, wherein the growth medium comprises a lava, cavaain extract and corn oil. 28. A method for killing molluscs, characterized in that a moluscocidal nematode of the genus Phasmarnabditis is applied to an area which is infested with molluscs, in conjunction with bacteria that promote their growth. Damage, snails (% - angular scale) Without treatment Met h i oca r b Nematode —-Φ— .- .. θ .... -